臺灣博碩士論文加值系統

樹突細胞對後天免疫系統的貢獻主要在於對T細胞的調控，並且被認為可藉由細胞表面的Notch ligands去活化T細胞上的Notch訊息傳遞以改變T細胞亞群的分化而有不同的免疫反應。已知益生菌能影響樹突細胞的成熟和免疫調節功能，並且可經由與樹突細胞表面不同的類鐸受器(TLRs)結合來影響，但是益生菌是否會藉由TLRs訊息傳遞來影響到樹突細胞Notch或Notch ligands的表現目前仍不清楚。實驗中先利用人類周邊血液單核球及樹突細胞之體外培養，與加熱失去活性的益生菌作用後觀察細胞激素的變化以了解各種Lactobacilli益生菌對於誘發人體免疫反應之效能，接著以小鼠骨髓衍生樹突細胞篩選出最具潛力的A菌，觀察此菌對於樹突細胞Notch、Notch ligands、Hes1、IL-10和IL-12等基因表現之影響，並在IL-10存在環境下觀察基因的表現是否有細胞激素的參與，再以gamma secretase inhibitor (GSI) 抑制Notch訊息傳遞後觀察樹突細胞成熟分子的表現是否受到影響，以及觀察餵食A菌之OVA致敏小鼠是否改善氣喘的症狀。結果顯示A菌能大幅提升小鼠骨髓衍生樹突細胞Delta1、Delta4、Jagged1、IL-12和Hes1 在24小時前期增加表現量，而Notch3和IL-10基因則在24小時後期能持續增加表現量，此一現象可能是藉由菌之細胞壁的peptidoglycan與樹突細胞上的TLR2以及菌之CpG DNA與TLR9作用而造成，TLR4也可能參與不同程度各基因的變化，同時IL-10則是降低Notch和Notch ligands基因的表現。另外，A菌刺激之樹突細胞能增加下游T細胞IFN-γ的分泌，同時藉由TLR2、TLR4、TLR9影響下游T細胞的增生，並且從GSI不改變樹突細胞成熟分子的表現可看出Notch基因的變化不影響樹突細胞本身之功能，而餵食A菌的小鼠能有效減緩以methacholine誘發之呼吸道阻力增加情形。從結果中推論益生菌能藉由TLR2、4、9改變樹突細胞早期Notch ligands表現量，引起Notch訊息傳遞再啟動Notch3基因的表現，輔以細胞激素之調節，並且可能參與樹突細胞對T細胞之影響走向Th1分化，以緩和過敏性氣喘的反應。

Notch ligands on dendritic cells initiate Notch signals on T cells to drive T cell differentiation and result in different immune responses in adaptive immunity. Probiotics are recognized by dendritic cells via Toll-like Receptor (TLR) 2, TLR4 or TLR9. Activating TLR signals effect on maturation and immune modulation on dendritic cells. Though there is studies on Nocth signals and TLRs recently, the relationship between probiotics and Notch signals is still unknown. Thus we used heat-inactivated probiotics to stimulate human peripheral blood mononuclear cells and monocyte-derived dendritic cells and investigated what kinds of immune responses were induced. Furthermore, we selected probiotic A as the most potential strain and investigated the gene expression of Notch, Notch ligands, Hes1, IL-10 and IL-12 on probiotic A–stimulated bone marrow-derived murine dendritic cells in vitro. We observed that probiotic A up-regulated Delta1, Delta4, Jagged1, Hes1 and IL-12 gene expression on dendritic cells in early phases between 3 to 24 hours while Notch3 and IL-10 gene were continuous increase after 24 hours stimulation. Moreover, IL-10 might reverse the changes. Our data suggest that probiotic A induced Notch/Notch ligands expression by TLR2 and TLR9 signaling in murine dendritic cells. However, TLR4 also involved these gene expression. In conclusion, it is possible that Notch, TLR and cytokine signals are integrated to modulate specific effector functions in dendritic cells and, therefore, induce the development of Th1 cells to ease the airway resistance in murine asthma model.

目錄
謝誌----------------------------------------------------------------------------------3
中文摘要----------------------------------------------------------------------------5
英文摘要----------------------------------------------------------------------------7
縮寫名詞對照表-------------------------------------------------------------------9
第一章 緒論----------------------------------------------------------------------10
(一) 益生菌
(二) 乳酸桿菌
(三) 免疫細胞的調控
(四) 益生菌和免疫細胞之間的關係
(五) Toll-like receptor
(六) Notch以及Notch配位子
(七) Notch以及Notch配位子與樹突細胞之間的關係
(八) Notch以及Notch配位子與T細胞之間的關係
第二章 研究動機與目的-------------------------------------------------------24
第三章 探討益生菌對人類免疫細胞功能之影響-------------------------27
(一) 實驗目的與原理
(二) 實驗材料與方法
(三) 實驗結果
(四) 討論
第四章 探討益生菌對小鼠免疫細胞功能之影響-------------------------42
(一) 實驗目的與原理
(二) 實驗材料與方法
(三) 實驗結果
(四) 討論

第五章 探討益生菌藉由Notch及Notch配位子對小鼠樹突細胞免疫功能之調控---------------------------------------------------------------------------50
(一) 實驗目的與原理
(二) 實驗材料與方法
(三) 實驗結果
(四) 討論
第六章 結論與未來方向-------------------------------------------------------71
第七章 圖表----------------------------------------------------------------------73
第八章 參考文獻----------------------------------------------------------------98

1.Lilly, D. M., and R. H. Stillwell. 1965. Probiotics: Growth-Promoting Factors Produced by Microorganisms. Science 147:747-748.
2.Fuller, R. 1989. Probiotics in man and animals. J Appl Bacteriol 66:365-378.
3.Tuomola, E. M., A. C. Ouwehand, and S. J. Salminen. 1999. Human ileostomy glycoproteins as a model for small intestinal mucus to investigate adhesion of probiotics. Lett Appl Microbiol 28:159-163.
4.Sheil, B., J. McCarthy, L. O''Mahony, M. W. Bennett, P. Ryan, J. J. Fitzgibbon, B. Kiely, J. K. Collins, and F. Shanahan. 2004. Is the mucosal route of administration essential for probiotic function? Subcutaneous administration is associated with attenuation of murine colitis and arthritis. Gut 53:694-700.
5.Forsythe, P., M. D. Inman, and J. Bienenstock. 2007. Oral treatment with live Lactobacillus reuteri inhibits the allergic airway response in mice. Am J Respir Crit Care Med 175:561-569.
6.Tsai, Y. T., P. C. Cheng, C. K. Fan, and T. M. Pan. 2008. Time-dependent persistence of enhanced immune response by a potential probiotic strain Lactobacillus paracasei subsp. paracasei NTU 101. Int J Food Microbiol.
7.Perdigon, G., E. Vintini, S. Alvarez, M. Medina, and M. Medici. 1999. Study of the possible mechanisms involved in the mucosal immune system activation by lactic acid bacteria. J Dairy Sci 82:1108-1114.
8.Torii, A., S. Torii, S. Fujiwara, H. Tanaka, N. Inagaki, and H. Nagai. 2007. Lactobacillus Acidophilus strain L-92 regulates the production of Th1 cytokine as well as Th2 cytokines. Allergol Int 56:293-301.
9.Chuang, L., K. G. Wu, C. Pai, P. S. Hsieh, J. J. Tsai, J. H. Yen, and M. Y. Lin. 2007. Heat-killed cells of lactobacilli skew the immune response toward T helper 1 polarization in mouse splenocytes and dendritic cell-treated T cells. J Agric Food Chem 55:11080-11086.
10.Kim, J. Y., Y. O. Choi, and G. E. Ji. 2008. Effect of oral probiotics (Bifidobacterium lactis AD011 and Lactobacillus acidophilus AD031) administration on ovalbumin-induced food allergy mouse model. J Microbiol Biotechnol 18:1393-1400.
11.Fink, L. N., and H. Frokiaer. 2008. Dendritic cells from Peyer''s patches and mesenteric lymph nodes differ from spleen dendritic cells in their response to commensal gut bacteria. Scand J Immunol 68:270-279.
12.Sunada, Y., S. Nakamura, and C. Kamei. 2008. Effect of Lactobacillus acidophilus strain L-55 on the development of atopic dermatitis-like skin lesions in NC/Nga mice. Int Immunopharmacol 8:1761-1766.
13.Sakaguchi, S. 2003. The origin of FOXP3-expressing CD4+ regulatory T cells: thymus or periphery. J Clin Invest 112:1310-1312.
14.Hori, S., T. Takahashi, and S. Sakaguchi. 2003. Control of autoimmunity by naturally arising regulatory CD4+ T cells. Adv Immunol 81:331-371.
15.Takahashi, T., and S. Sakaguchi. 2003. Naturally arising CD25+CD4+ regulatory T cells in maintaining immunologic self-tolerance and preventing autoimmune disease. Curr Mol Med 3:693-706.
16.Sakaguchi, S., S. Hori, Y. Fukui, T. Sasazuki, N. Sakaguchi, and T. Takahashi. 2003. Thymic generation and selection of CD25+CD4+ regulatory T cells: implications of their broad repertoire and high self-reactivity for the maintenance of immunological self-tolerance. Novartis Found Symp 252:6-16; discussion 16-23, 106-114.
17.Larche, M. 2007. Regulatory T cells in allergy and asthma. Chest 132:1007-1014.
18.Akdis, C. A., K. Blaser, and M. Akdis. 2004. Genes of tolerance. Allergy 59:897-913.
19.Perdigon, G., S. Alvarez, M. Medina, E. Vintini, and E. Roux. 1999. Influence of the oral administration of lactic acid bacteria on iga producing cells associated to bronchus. Int J Immunopathol Pharmacol 12:97-102.
20.Lee, J., J. H. Mo, C. Shen, A. N. Rucker, and E. Raz. 2007. Toll-like receptor signaling in intestinal epithelial cells contributes to colonic homoeostasis. Curr Opin Gastroenterol 23:27-31.
21.Rakoff-Nahoum, S., L. Hao, and R. Medzhitov. 2006. Role of toll-like receptors in spontaneous commensal-dependent colitis. Immunity 25:319-329.
22.Chieppa, M., M. Rescigno, A. Y. Huang, and R. N. Germain. 2006. Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement. J Exp Med 203:2841-2852.
23.Steinman, R. M., D. Hawiger, and M. C. Nussenzweig. 2003. Tolerogenic dendritic cells. Annu Rev Immunol 21:685-711.
24.Kanzato, H., S. Fujiwara, W. Ise, S. Kaminogawa, R. Sato, and S. Hachimura. 2008. Lactobacillus acidophilus strain L-92 induces apoptosis of antigen-stimulated T cells by modulating dendritic cell function. Immunobiology 213:399-408.
25.Akira, S., and K. Takeda. 2004. Toll-like receptor signalling. Nat Rev Immunol 4:499-511.
26.Latz, E., A. Schoenemeyer, A. Visintin, K. A. Fitzgerald, B. G. Monks, C. F. Knetter, E. Lien, N. J. Nilsen, T. Espevik, and D. T. Golenbock. 2004. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol 5:190-198.
27.Latz, E., A. Visintin, T. Espevik, and D. T. Golenbock. 2004. Mechanisms of TLR9 activation. J Endotoxin Res 10:406-412.
28.Tsukumo, S., and K. Yasutomo. 2004. Notch governing mature T cell differentiation. J Immunol 173:7109-7113.
29.Hu, X., A. Y. Chung, I. Wu, J. Foldi, J. Chen, J. D. Ji, T. Tateya, Y. J. Kang, J. Han, M. Gessler, R. Kageyama, and L. B. Ivashkiv. 2008. Integrated regulation of Toll-like receptor responses by Notch and interferon-gamma pathways. Immunity 29:691-703.
30.Varnum-Finney, B., L. Xu, C. Brashem-Stein, C. Nourigat, D. Flowers, S. Bakkour, W. S. Pear, and I. D. Bernstein. 2000. Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nat Med 6:1278-1281.
31.Yamaguchi, E., S. Chiba, K. Kumano, A. Kunisato, T. Takahashi, and H. Hirai. 2002. Expression of Notch ligands, Jagged1, 2 and Delta1 in antigen presenting cells in mice. Immunol Lett 81:59-64.
32.Yvon, E. S., S. Vigouroux, R. F. Rousseau, E. Biagi, P. Amrolia, G. Dotti, H. J. Wagner, and M. K. Brenner. 2003. Overexpression of the Notch ligand, Jagged-1, induces alloantigen-specific human regulatory T cells. Blood 102:3815-3821.
33.Olivier, A., E. Lauret, P. Gonin, and A. Galy. 2006. The Notch ligand delta-1 is a hematopoietic development cofactor for plasmacytoid dendritic cells. Blood 107:2694-2701.
34.Napolitani, G., A. Rinaldi, F. Bertoni, F. Sallusto, and A. Lanzavecchia. 2005. Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells. Nat Immunol 6:769-776.
35.Cheng, P., A. Zlobin, V. Volgina, S. Gottipati, B. Osborne, E. J. Simel, L. Miele, and D. I. Gabrilovich. 2001. Notch-1 regulates NF-kappaB activity in hemopoietic progenitor cells. J Immunol 167:4458-4467.
36.Amsen, D., J. M. Blander, G. R. Lee, K. Tanigaki, T. Honjo, and R. A. Flavell. 2004. Instruction of distinct CD4 T helper cell fates by different notch ligands on antigen-presenting cells. Cell 117:515-526.
37.Wakui, M., K. Nakano, and S. Matsushita. 2007. Notch ligand mRNA levels of human APCs predict Th1/Th2-promoting activities. Biochem Biophys Res Commun 358:596-601.
38.Maekawa, Y., S. Tsukumo, S. Chiba, H. Hirai, Y. Hayashi, H. Okada, K. Kishihara, and K. Yasutomo. 2003. Delta1-Notch3 interactions bias the functional differentiation of activated CD4+ T cells. Immunity 19:549-559.
39.Glimcher, L. H. 2001. Lineage commitment in lymphocytes: controlling the immune response. J Clin Invest 108:s25-s30.
40.Pui, J. C., D. Allman, L. Xu, S. DeRocco, F. G. Karnell, S. Bakkour, J. Y. Lee, T. Kadesch, R. R. Hardy, J. C. Aster, and W. S. Pear. 1999. Notch1 expression in early lymphopoiesis influences B versus T lineage determination. Immunity 11:299-308.
41.Eagar, T. N., Q. Tang, M. Wolfe, Y. He, W. S. Pear, and J. A. Bluestone. 2004. Notch 1 signaling regulates peripheral T cell activation. Immunity 20:407-415.
42.Amsen, D., A. Antov, D. Jankovic, A. Sher, F. Radtke, A. Souabni, M. Busslinger, B. McCright, T. Gridley, and R. A. Flavell. 2007. Direct regulation of Gata3 expression determines the T helper differentiation potential of Notch. Immunity 27:89-99.
43.Debarry, J., H. Garn, A. Hanuszkiewicz, N. Dickgreber, N. Blumer, E. von Mutius, A. Bufe, S. Gatermann, H. Renz, O. Holst, and H. Heine. 2007. Acinetobacter lwoffii and Lactococcus lactis strains isolated from farm cowsheds possess strong allergy-protective properties. J Allergy Clin Immunol 119:1514-1521.
44.Ichikawa, S., R. Fujii, D. Fujiwara, Y. Komiyama, T. Kaisho, M. Sakaguchi, and Y. Konishi. 2007. MyD88 but not TLR2, 4 or 9 is essential for IL-12 induction by lactic acid bacteria. Biosci Biotechnol Biochem 71:3026-3032.
45.Palaga, T., C. Buranaruk, S. Rengpipat, A. H. Fauq, T. E. Golde, S. H. Kaufmann, and B. A. Osborne. 2008. Notch signaling is activated by TLR stimulation and regulates macrophage functions. Eur J Immunol 38:174-183.
46.Pochard, P., P. Gosset, C. Grangette, C. Andre, A. B. Tonnel, J. Pestel, and A. Mercenier. 2002. Lactic acid bacteria inhibit TH2 cytokine production by mononuclear cells from allergic patients. J Allergy Clin Immunol 110:617-623.
47.Niers, L. E., H. M. Timmerman, G. T. Rijkers, G. M. van Bleek, N. O. van Uden, E. F. Knol, M. L. Kapsenberg, J. L. Kimpen, and M. O. Hoekstra. 2005. Identification of strong interleukin-10 inducing lactic acid bacteria which down-regulate T helper type 2 cytokines. Clin Exp Allergy 35:1481-1489.
48.Foligne, B., S. Nutten, C. Grangette, V. Dennin, D. Goudercourt, S. Poiret, J. Dewulf, D. Brassart, A. Mercenier, and B. Pot. 2007. Correlation between in vitro and in vivo immunomodulatory properties of lactic acid bacteria. World J Gastroenterol 13:236-243.
49.Fiorentino, D. F., A. Zlotnik, T. R. Mosmann, M. Howard, and A. O''Garra. 1991. IL-10 inhibits cytokine production by activated macrophages. J Immunol 147:3815-3822.
50.Ongol, M. P., T. Iguchi, M. Tanaka, T. Sone, H. Ikeda, K. Asano, and T. Nishimura. 2008. Potential of selected strains of lactic Acid bacteria to induce a Th1 immune profile. Biosci Biotechnol Biochem 72:2847-2857.
51.Rothenfusser, S., E. Tuma, S. Endres, and G. Hartmann. 2002. Plasmacytoid dendritic cells: the key to CpG. Hum Immunol 63:1111-1119.
52.Aliprantis, A. O., R. B. Yang, M. R. Mark, S. Suggett, B. Devaux, J. D. Radolf, G. R. Klimpel, P. Godowski, and A. Zychlinsky. 1999. Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science 285:736-739.
53.Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda, and S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11:443-451.
54.Schwandner, R., R. Dziarski, H. Wesche, M. Rothe, and C. J. Kirschning. 1999. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J Biol Chem 274:17406-17409.
55.Cox, K. H., I. Ofek, and D. L. Hasty. 2007. Enhancement of macrophage stimulation by lipoteichoic acid and the costimulant hemoglobin is dependent on Toll-like receptors 2 and 4. Infect Immun 75:2638-2641.
56.Traub, S., S. von Aulock, T. Hartung, and C. Hermann. 2006. MDP and other muropeptides--direct and synergistic effects on the immune system. J Endotoxin Res 12:69-85.
57.Strober, W., P. J. Murray, A. Kitani, and T. Watanabe. 2006. Signalling pathways and molecular interactions of NOD1 and NOD2. Nat Rev Immunol 6:9-20.
58.Amsen, D., A. Antov, and R. A. Flavell. 2009. The different faces of Notch in T-helper-cell differentiation. Nat Rev Immunol 9:116-124.
59.Steinbrink, K., H. Jonuleit, G. Muller, G. Schuler, J. Knop, and A. H. Enk. 1999. Interleukin-10-treated human dendritic cells induce a melanoma-antigen-specific anergy in CD8(+) T cells resulting in a failure to lyse tumor cells. Blood 93:1634-1642.
60.Steinbrink, K., E. Graulich, S. Kubsch, J. Knop, and A. H. Enk. 2002. CD4(+) and CD8(+) anergic T cells induced by interleukin-10-treated human dendritic cells display antigen-specific suppressor activity. Blood 99:2468-2476.
61.Jonuleit, H., E. Schmitt, K. Steinbrink, and A. H. Enk. 2001. Dendritic cells as a tool to induce anergic and regulatory T cells. Trends Immunol 22:394-400.